JP2007520233A - Compositions and methods for culturing neural stem cells using bone marrow stromal cells - Google Patents

Abstract

  The present invention includes methods and compositions for promoting proliferation of neural stem cells. A method for regulating the expression of MHC molecules in neural stem cells (NSC) is also included in the present invention.

Description

  The bone marrow includes at least two types of stem cells, namely hematopoietic stem cells, as well as non-hematopoietic tissue stem cells, variously called mesenchymal stem cells, myeloid stem cells (MSC) or bone marrow stromal cells (BMSC). In this specification, these terms are used synonymously. MSCs are interesting because they are easily separated with a small amount of aspiration of the bone marrow and easily form colonies derived from single cells. A single cell-derived colony can grow after 50 doublings in about 10 weeks, and osteoblasts, adipocytes, chondrocytes (Non-patent Document 1; Non-patent Document 2; Non-patent Document) 3; Non-patent document 4), myocytes (non-patent document 5), astrocytes, oligodendrocytes (oligodendrocytes), and neurons (non-patent document 6; non-patent document 7; non-patent document 8) Can be differentiated into non-patent document 9).

  Furthermore, MSC generates cells of all three germ layers (Non-patent document 7; Non-patent document 10; Non-patent document 11; Non-patent document 12; Non-patent document 13). In vivo experiments have shown that not only pure MSC cells but also bone marrow-derived non-dividing cells generate epithelial cell types including lung epithelial cells (Non-Patent Document 14; Non-Patent Documents) 15) In addition, several recent studies have shown that transplantation of MSC is promoted by tissue damage (Non-Patent Document 16; Non-Patent Document 17). For these reasons, the possibility that MSC can be used for cell therapy and gene therapy for many human diseases is currently being investigated (Non-patent Document 18; Non-patent Document 19).

  MSC is an alternative source of pluripotent stem cells. Under physiological conditions, they maintain the structure of the bone marrow with various cell adhesion molecules and regulate hematopoiesis with secretions consisting of cytokines (20). MSCs grown from bone marrow can be efficiently propagated by selective attachment to tissue culture plastic (Non-patent document 6; Non-patent document 21), and can be genetically manipulated (non-patent document 22). .

  In addition, MSC can differentiate into various mesodermal tissues including bone (Non-patent Document 23), cartilage (Non-patent Document 24), fat (Non-patent Document 23), and muscle (Non-patent Document 5). Also called mesenchymal stem cells. Furthermore, it has been reported that the cells differentiate into nerve-like cells expressing a neuronal marker (Non-patent Document 9; Non-patent Document 25; Non-patent Document 26), and therefore MSC may overcome the restriction of germ layers. Has been suggested.

  Stem cells are pluripotent progenitor cells that have a wide range of developmental potential in a predetermined tissue and a predetermined time, and self-regenerate (Non-patent Document 27). Much interest has recently emerged from studies of stem cells of the nervous system, not only because of the importance of understanding the development of the nervous system, but also because of its therapeutic potential in the treatment of neurodegenerative diseases.

  Current methods for isolating and maintaining embryonic stem cells, and other stem cell lines, rely on the use of murine fetal fibroblasts (MEF) as a feeder cell layer. It has been reported that the frequency of embryonic stem cell clones increases several times when a serum substitute is used in the culture medium (Non-patent Document 28). The presence of basic fibroblast growth factor (bFGF) is essential for the growth of cloned embryonic stem cells in an undifferentiated state. Current methods for isolation, culture, and expansion of human embryonic stem cells are limited due to their dependence on the murine fetal fibroblast feeder cell layer. Also, it has not been demonstrated that stem cells can be maintained indefinitely indefinitely in the absence of feeder cells.

  Several different methods and growth factors (GF) have been used over the last decade by many different groups to improve the growth rate of human fetal brain stem cells. bFGF and epidermal growth factor (EGF) have been shown to be necessary for the growth and maintenance of human embryonic neural stem cells (hNSC). Usually, these human NSC cultures are grown as free floating cell clusters (neurospheres), but neurospheres cannot be grown indefinitely with bFGF and EGF alone. Leukemia inhibitory factor (LIF) has been shown to increase the growth rate of FGF and EGF-reactive NSCs and prolong lifespan (Carpenter et al., 1999 and Wright et al., 2003). In addition to controlling the growth rate, LIF dynamically controls several genes, including NSC's major histocompatibility complex (MHC) molecules (Wright et al., 2003).

  Human fetal brain stem cells are considered as an attractive candidate for stem cell transplantation for regeneration of damaged tissue. Stem cells from embryos or fetal donors require allogeneic transplantation. Cell transplantation between genetically different individuals always carries the risk of graft rejection by the host. Most cells express the major histocompatibility complex, the product of MHC class I molecules. Furthermore, many cell types are susceptible to the expression of MHC class II molecules when exposed to inflammatory cytokines. Allograft rejection is mediated primarily by both CD4 and CD8 subclass T cells (Non-patent Document 29). Alloreactive CD4 T cells produce cytokines that exacerbate the cytolytic CD8 response to alloantigens. Within these subclasses, competitive subpopulation cells develop after antigen stimulation, which is characterized by the cytokines they produce. Th1 cells producing IL-2 and IFN-γ are mainly involved in allograft rejection (Non-patent Document 30). Th2 cells producing IL-4 and IL-10 can down-regulate the Th1 response via IL-10 (Non-patent Document 31). In fact, much effort has been expended to avoid the unwanted Th1 response and move to the Th2 pathway. Undesirable alloreactive T cell responses to patient grafts are generally treated with immunosuppressive agents such as prednisone, azathioprine, cyclosporin A. Unfortunately, these drugs generally need to be administered for the patient's life, but have many dangerous side effects, including systemic immunosuppression.

Neural stem cells express low or negligible levels of MHC class I and / or class II antigens (Non-Patent Document 32), but usually these cells are allogeneic unless an immunosuppressant is used. Rejected after transplant to recipient. Presumably, MHC molecules are upregulated on the cell membrane after exposure to inflammatory cytokines of the IFN family, after which rejection is initiated.
AJ Freidenstein et al., 1970 Cell Tissue Kinet. 3: 393-403 H. Castro-Malaspina, 1980 Blood 56: 289-301 NN Beresford et al., 1992 J. Cell Sci. 102: 341-351 DJ PROCUP 1997 Science 276: 71-74 S. Wakitani et al., 1995 Muscle Nerve 18: 1417-1426 SA Ajiji et al., 1998 Proc. Natl. Acad. Sci. USA 95: 3908-3913 GC Copen et al., 1999 Proc. Natl. Acad. Sci. USA 96: 10711-10716 M. Chop et al., 2000 NeuroreportII, 3001-3005 D. Woodbury et al., 2000 Neuroscience Res. 61: 364-370 Litchi, KW et al., 2000 Nature Med. 6: 1282-1286 Cotton, DN, etc., 2001 Development 128: 5181-5188 Toma, C et al., 2002 Circulation 105: 93-98 Yang, Y et al., 2002 Nature 418: 41-49 Cloudy et al., 2001 Cell 105: 369-377 Petersen et al., 1999 Science 284: 1168-1170 Ferrari, G et al., 1998 Science 279: 1528-1530 Okamoto, R et al., 2002 Nature Med. 8: 1101-1017 Horowitz et al., 1999 Nat. Med. 5: 309-313 Caplan et al., 2000 Clin. Orthoped. 379: 567-570 Clark, BR and Keating, A. 1995 Ann NY Acad Sci 770: 70-78 Coulter, D. C et al., 2000 Proc Natl Acad Sci USA 97: 3213-218 Schwartz, EJ et al., 1999 Hum Gene Ther 10: 2539-2549 Beresford, J. N et al., 1992 J Cell Sci 102: 341-351 Lennon, D.P et al., 1995 Exp Cell Res 219: 211-222 Sanchez-Ramos, J et al., 2000 Exp Neurol 164: 247-256 Den, W et al., 2001 Biochem Biophys Res Commun 282: 148-152 Morrison et al., 1997 Cell 88: 287-298 Amit et al., 2000 Dev Biol 227: 271-278 Rosenberg et al., 1992 Annu. Rev. Immunol. 10: 333 Mossman et al., 1989 Annu. Rev. Immunol. 7: 145 Fiorentino et al., 1989 J. Exp. Med. 170: 2081 McLaren et al., 2001 J. Neuroimmunol. 112: 35

  Therefore, standardization of culture conditions that maximize NSC growth for therapy and NSC pluripotency are highly needed. Furthermore, it is now believed that the success of NSC transplantation is due to the prevention and / or reduction of undesirable immune responses to NSCs mediated by immune effectors to prevent NSC host rejection. That is, there has long been a need for a method of suppressing or preventing undesirable immune responses associated with NSC transplantation between genetically different individuals. The present invention satisfies this need.

  The present invention includes compositions and methods for culturing neural stem cells (NSC). The invention also includes cells produced by such compositions and methods.

  The present invention includes a composition comprising an isolated bone marrow stromal cell (BMSC) and a neural stem cell (NSC) growth medium and a chemically defined medium containing factors secreted by the BMSC.

  According to one embodiment, the medium does not contain exogenous leukemia inhibitory factor (LIF).

  According to another embodiment, the factor secreted by BMSC is selected from the group consisting of growth factors, trophic factors, and cytokines.

  According to yet another embodiment, the factor is LIF, brain-derived neurotrophic factor (BDNF), epidermal growth factor receptor (EGF), basic fibroblast growth factor (bFGF), FGF-6, glial cell-derived nerve Nutritional factor (GDNF), granulocyte colony stimulating factor (GCSF), hepatocyte growth factor (HGF), IFN-γ, insulin-like growth factor binding protein (IGFBP-2), IGFBP-6, IL-1ra, IL-6 , IL-8, monocyte chemotactic protein (MCP-1), mononuclear phagocyte colony stimulating factor (M-CSF), neurotrophic factor (NT3), tissue metalloprotease inhibitor (TIMP-1), TIMP-2, tumor necrosis factor (TNF-β), vascular endothelial growth factor (VEGF), VEGF-D, urokinase plasminogen activator receptor (uPAR), bone morphogenetic protein 4 (BMP4), IL1-a , IL-3, leptin, stem cell factor (SCF), stromal cell-derived factor-1 (SDF-1), platelet-derived growth factor-BB (PDGFBB), tumor cell growth factor beta (TGFβ-1), and TGFβ-3 Selected from the group consisting of Is done.

  The present invention also includes co-culturing BMSCs with NSCs in a chemically defined medium containing neural stem cell (NSC) growth medium.

  According to one embodiment, the BMSC and NSC are cultured in a contact-dependent manner in which the BMSC is in physical contact with the NSC.

  According to another embodiment, the BMSC and NSC are cultured in a contact-independent manner where the BMSC is not in physical contact with the NSC.

  According to a further embodiment, the NSC is derived from the human central nervous system.

  According to yet another embodiment, the BMSC is human.

  According to another embodiment, exogenous genetic material has been introduced into the cells of the invention.

  The invention also encompasses a bone marrow stromal cell conditioned medium (BMSC-CM) comprising a neural stem cell (NSC) growth medium and a chemically defined medium containing factors secreted by the isolated BMSC.

  According to one aspect, the BMSC-CM does not include exogenous LIF.

  According to another embodiment, BMSC is essentially absent from the BMSC-CM.

  Furthermore, according to another aspect of the present invention, the BMSC-CM comprises a factor selected from the group consisting of a growth factor, a nutrient factor, and a cytokine.

  According to yet another embodiment, BMSC-CM is derived from LIF, brain-derived neurotrophic factor (BDNF), epidermal growth factor receptor (EGF), basic fibroblast growth factor (bFGF), FGF-6, glial cells Neurotrophic factor (GDNF), granulocyte colony stimulating factor (GCSF), hepatocyte growth factor (HGF), IFN-γ, insulin-like growth factor binding protein (IGFBP-2), IGFBP-6, IL-1ra, IL- 6, IL-8, monocyte chemotactic protein (MCP-1), mononuclear phagocyte colony stimulating factor (M-CSF), neurotrophic factor (NT3), tissue metalloprotease inhibitor (TIMP-1) , TIMP-2, tumor necrosis factor (TNF-β), vascular endothelial growth factor (VEGF), VEGF-D, urokinase plasminogen activator receptor (uPAR), bone morphogenetic protein 4 (BMP4), IL1- a, IL-3, leptin, stem cell factor (SCF), stromal cell-derived factor-1 (SDF-1), platelet-derived growth factor-BB (PDGFBB), tumor cell growth factor beta (TGFβ-1), and TGFβ- Select from the group consisting of 3 It is.

  The invention also encompasses methods for modulating the expression of major histocompatibility complex (MHC) molecules in isolated NSCs.

  According to one aspect, co-culture of isolated NSCs and isolated BMSCs regulates the expression of MHC molecules in the NSCs.

  According to another embodiment, the expression of MHC molecules in NSCs can be regulated by culturing NSCs with BMSC-CM.

  The present invention encompasses isolated NSCs prepared from BMSC and NSC co-cultures.

  According to one aspect of the present invention, NSCs produced by the methods of the present invention exhibit reduced expression of MHC class I molecules.

  According to another embodiment, NSCs produced by the methods of the present invention exhibit baseline levels of MHC class II molecules.

  The present invention also includes a nerve cell culture apparatus including isolated NSC, isolated BMSC, NSC growth medium, and means for preventing the NSC and the BMSC from being in physical contact with each other. .

  According to another aspect, the device further comprises a filter or membrane to prevent NSC and BMSC from coming into physical contact with each other.

  According to another embodiment, the filter or membrane has pores that allow factors secreted from the BMSC to permeate the filter or membrane.

  For the purpose of illustrating the invention, certain embodiments of the invention are shown in the accompanying drawings. However, the present invention is not limited to the apparatus and means of the embodiments shown in the figures.

Figure 1 shows BMSC medium (DMEM-low glucose, 10% lot tested fetal bovine serum (FBS)) or NSC medium (DMEM-F12, N2 added, EGF 20 ng / ml, bFGFlOng / ml, heparin 8 μg / ml, penicillin -Is a graph showing the proliferation of BMSCs with streptomycin (P / S).
2A-2C are a series of images showing NSCs growing in a neurosphere shape. Figures 2B and 2C show NSCs grown in the presence of exogenous leukemia inhibitory factor (LIF). FIG. 2A shows neurospheres growing in the absence of exogenous LIF.
3A-3D are a series of images showing BMSC and NSC co-culture in the presence of exogenous LIF (FIGS. 3C and 3D) and in the absence (FIGS. 3A and 3B).
4A-4H are a series of pre-differentiation images showing nestin and glial fibrillary acidic protein (GFAP) expression by NSCs in co-culture with BMSC (FIGS. 4A-4F). Figures 4G and 4H show the absence of nestin and GFAP by BMSC cultured alone.
FIGS. 5A to 5D are a series of images showing that NSCs grown on BMSC possess the ability to differentiate into neurons and astrocytes. Figures 5A-5D show MAP2-GFAP-DAPI staining of differentiated co-cultures. MAP2 is a cytoskeletal protein. DAPI is 4 ', 6'-diamino-2-phenylindole hydrochloride that stains nuclei.
FIG. 6 is an image showing nestin-GFAP staining of differentiated co-cultures showing negligible nestin expression after differentiation.
FIG. 7 is an image showing MAP2-GFAP staining of differentiated NSCs.
Figures 8A and 8B are a series of images showing MAP2-GFAP-DAPI staining of differentiated BMSCs showing negligible expression of neuronal and astrocyte markers.
9A and 9B are a series of FACS analysis graphs showing the phenotype of NSC after removal of BMSC. FIG. 9A shows that less than 2% of the cells are CD-105 (BMSC marker) positive. FIG. 9B shows that over 90% of the cells are CD133 positive.
FIG. 10A is a series of FACS analysis graphs showing expression in the absence of nestin by BMSC. FIG. 10B is a series of FACS analysis graphs showing nestin expression by NSCs isolated from co-cultures using BMSC feeder cells.
FIG. 11 is an image showing that NSCs proliferating on BMSC possess pluripotency to differentiate into neurons and astrocytes.
FIG. 12 is a graph showing the growth of NSCs in Transwell (registered trademark) in the presence or absence of BMSC.
FIG. 13A is an image showing NSC grown on an uncoated plate in the presence of BMSC in Transwell ™. FIG. 13B is an image showing NSC grown on an uncoated plate in the absence of BMSC in Transwell ™.
FIG. 14A is an image showing NSCs cultured using bone marrow stromal cell conditioned medium (BMSC-CM). FIG. 14B is an image showing NSCs cultured using bone marrow stromal cell conditioned medium (BMSC-CM). FIG. 14C is an image showing NSCs cultured in NSC medium in the presence of exogenous LIF. FIG. 14D is an image showing NSCs cultured in NSC medium in the absence of exogenous LIF.
FIG. 15 is a graph showing the effect of BMSC-CM compared to standard NSC medium with or without exogenous LIF on NSC proliferation. BMSC-CM1 is a medium by BMSC cultured in an NSC medium in which EGF and FGF are present. BMSC-CM2 is a medium by BMSC cultured in NSC medium without EGF and FGF FIGS. 16A-16D show the phenotypic profiles of NSCs isolated from co-cultures using BMSCs from two different donors FIG. 16 is a series of FACS analysis graphs (FIGS. 16A and 16B). FIGS. 16C and 16D are a series of graphs showing phenotypic profiles of NSCs cultured in NSC medium without BMSC or NSC medium with exogenous LIF, respectively.
17A-17D are a series showing the phenotypes of NSCs cultured in NSC medium in the presence and absence of exogenous LIF, and NSCs cultured in BMSC-CM in the presence and absence of growth factors. It is a FACS analysis graph. Figures 17A through 17D show the profiles of CD56, CD133, MHC class II molecules, and MHC class I molecules, respectively.
18A-18D show the phenotype of NSC cultured in NSC medium in the presence (FIG. 18B) and absence (FIG. 18A) of exogenous LIF, and the phenotype of NSC co-cultured with BMSC in complete NSC medium. Figure 19 is a series of FACS analysis graphs showing (Figures 18C and 18D). FIG. 18 shows the expression of MHC class I and class II molecules by NSCs cultured under various conditions (black: isotype control; gray: class I or class II).

  The present invention includes compositions and methods for inducing and / or promoting neural stem cell (NSC) proliferation while maintaining its pluripotency. The invention also encompasses compositions and methods for modulating the expression of MHC molecules by NSCs.

  The present invention relates to the discovery that bone marrow stromal cells (BMSCs) can serve as feeder cell layers to support NSC proliferation. Thus, the present invention includes compositions and methods for using BMSC as a feeder cell layer for culturing NSCs and maintaining their pluripotency as well as to induce and / or promote NSC proliferation.

  In addition, in neural stem cell culture medium (NSC medium) supplemented with exogenous LIF to improve proliferation, compared to MHC molecule expression by NSC cultured in the absence of BMSC support cell layer, on BMSC support cell layer The present disclosure shows that up-regulation of MHC molecule expression and / or induction of expression by NSCs co-cultured with is reduced. That is, the present invention includes compositions and methods for reducing and / or preventing expression of MHC molecules by NSCs using BMSC as a feeder cell layer for NSC culture and proliferation.

  The data disclosed herein further indicate that BMSCs secrete growth factors, trophic factors, and / or cytokines that are useful for NSCs to grow while maintaining pluripotency. Factors secreted by BMSC can be obtained by culturing BMSCs in a medium within a certain period of time and then recovering conditioned medium for use thereafter. Accordingly, the present invention also includes bone marrow stromal cell conditioned medium (BMSC-CM) that is useful for growth while maintaining NSC pluripotency.

  The present invention also relates to the discovery that BMSC secretion factors present in BMSC-CM reduce MHC molecule expression up-regulation and / or expression induction by NSC. Thus, the present invention includes compositions and methods for growing NSCs using BMSC-CM in the absence and / or reduction of upregulation of MHC molecule expression on NSCs.

  Accordingly, the present invention includes methods and compositions for producing NSCs useful for therapy. That is, the present invention includes compositions and methods for producing NSCs useful for treating patients suffering from central nervous system diseases, disorders, or symptoms. The method includes culturing and expanding NSC and administering NSC to the patient.

(Definition)
As used herein, the following terms have the meanings used in this section.

  As used herein, the article “a” and “an” indicates that the grammatical object of the article is one or more than one (ie, at least one). As an example, “an element” means one element or more than one element.

  The term “about” will vary to some extent in the context in which it will be appreciated by one of ordinary skill in the art.

  The term “self” as used herein refers to any substance of the same origin as the individual being reintroduced.

  As used herein, the term “homologous” refers to any substance from a different individual of the same species.

  As used herein, the terms “bone marrow stromal cell”, “stromal cell”, “mesenchymal stem cell” or “MSC” are used interchangeably and stem cell-like progenitors of bone cells, chondrocytes and adipocytes. It refers to a small fraction of cells in the bone marrow that can act as a body and is isolated from the bone marrow by its ability to adhere to plastic dishes. Bone marrow stromal cells can be from any animal. In some embodiments, the stromal cells are primate, preferably human.

  As used herein, “differentiated” is the end of maturation such that the cells are fully mature and / or exhibit biological specialization and / or adaptation to a particular environment and / or function. A cell that has reached a stage. Normally, differentiated cells are characterized by the expression of genes encoding differentiation-related proteins in a given cell. For example, the expression of myelin protein and myelin sheath structure in glial cells is a typical example of terminally differentiated glial cells. When the term “differentiated” is used herein, the cell is in the process of differentiation.

  As used herein, “differentiation medium” refers to a cell growth medium that contains or lacks additives, in which the stem cells, embryonic stem cells, ES-like cells, neurospheres, NSCs or other similar progenitor cells that are not fully differentiated when cultured in medium mature to cells that have some or all of the characteristics of differentiated cells.

  “Proliferative” as used herein refers to the ability of a cell to proliferate, eg, when the number increases or a cell population doubles.

  The “supporting cell layer” is mediated by growth factors, cytokines, other cell-derived products, and inevitable contact in co-cultures to promote proliferation and maintain undifferentiated pluripotent stem cells. Means a cell that makes a physical support.

  As used herein, "supporting cells" refers to cells of a first tissue type that are co-cultured with cells of a second tissue type and provide an environment in which cells of the second tissue type can grow.

  The term “growth medium” as used herein refers to a medium that promotes cell growth. In general, the growth medium contains animal serum. In some cases, the growth medium does not contain animal serum.

  The term “NSC medium” as used herein refers to a medium for NSC culture and growth. Usually, NSC medium contains DMEM / F12, N2 supplement, EGF, bFGF, and heparin. In some cases, the NSC medium does not include growth factors (eg, EGF and bFGF).

  As used herein, “bone marrow stromal cell conditioned medium” (BMSC-CM) refers to a medium prepared by culturing BMSC. Based on the present disclosure, BMSC-CM is obtained by culturing BMSC in NSC medium, which medium has BMSC secreted growth factors, trophic factors, and cytokines in NSC medium along with other compounds. It was prepared using BMSC by culture. BMSC-CM can be used for NSC culture that promotes NSC proliferation while maintaining NSC pluripotency. Furthermore, BMSC-CM can be used for NSC culture that can regulate MHC molecule expression.

  As used herein, “leukemia inhibitory factor” (LIF) refers to a 22 kDa protein member of the interleukin-6 cytokine family that has multiple biological functions. LIF has been shown to induce terminal differentiation in leukemic cells, induce hematopoietic differentiation in normal and myeloid leukemia cells, and have the ability to stimulate acute phase protein synthesis in hepatocytes. Here, LIF was shown to promote the proliferation of undifferentiated NSCs while maintaining the pluripotency of NSCs.

  “Exogenous LIF” refers to LIF derived or produced from an external tissue, cell, or system.

  As used herein, the term “potential pluripotent” or “multipotent” refers to the ability of a central nervous system stem cell to differentiate into one or more types of cells. For example, pluripotent stem cells of the central nervous system can differentiate into neurons, astrocytes, and oligodendrocytes, without being limited thereto.

  “Neurosphere” as used herein refers to neural stem / progenitor cells whose nestin expression can be detected, particularly by immunostaining to detect nestin protein in the cells. Neurospheres are aggregates of proliferating neural stem cells, and the neurosphere structure is characteristic of neural stem cells in in vitro culture.

  As used herein, “neural stem cells” refers to undifferentiated, multipotent, self-replicating neurons. Neural stem cells are differentiable clonogenic pluripotent stem cells that have the ability to self-replicate under appropriate conditions and can ultimately differentiate into neurons, astrocytes, and oligodendrocytes. Therefore, neural stem cells are “multipotent” because there are multiple differentiation pathways in the progeny of stem cells. Neural stem cells can self-preserve in the sense that at each cell division, one daughter cell also becomes an average stem cell.

  As used herein, “neural cells” refers to cells that exhibit morphology, function, and phenotypic characteristics similar to those of glial cells and neurons from the central nervous system and / or peripheral nervous system. .

  As used herein, a “neuron-like cell” is a neuron-like marker that exhibits a morphology similar to that of a neuron and is detectable to a neuron-specific marker, such as, but not limited to, MAP2, neurofilament 200 kDa, neurofilament A cell that expresses -L, neurofilament-M, synaptophysin, β-tubulin III (TUJI), Tau, NeuN, neurofilament protein, and synaptic protein.

  As used herein, an “astrocyte-like cell” refers to a phenotype similar to that of an astrocyte, and a cell that expresses an astrocyte-specific marker such as, but not limited to, GFAP. To tell.

  As used herein, “oligodendrocyte-like cells” exhibit a phenotype similar to oligodendrocytes and express oligodendrocyte-specific markers such as, but not limited to, O-4. Say cell.

  As used herein, “cell cycle progression, or progression through the cell cycle” refers to the process by which a cell prepares for mitosis and / or meiosis and / or enters division. Progression through the cell cycle includes progression through the G1, S, G2, and M phases.

  “Proliferation” refers to a similar form of replication or multiplication, particularly for cells. That is, proliferation is the production of many cells, and can be measured in particular by simple calculation of the number of cells, measurement of 3H-thymidine incorporation into cells, and the like.

  The term “exogenous” as used herein refers to material introduced or produced by an external tissue, cell, or system.

  `` Encoding '' refers to any defined nucleotide sequence (i.e., rRNA, tRNA and mRNA), or a defined amino acid sequence, and a biological process that has the resulting biological properties. Refers to the unique properties of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, that serve as a template for the synthesis of other polymers and macromolecules. That is, in the case of mRNA transcription and translation in a cell or other biological system, the gene encodes a protein corresponding to the gene that makes the protein. The nucleotide sequence identical to the mRNA sequence, usually both the coding strand provided in the sequence listing and the non-coding strand used as a template for transcription of the gene or cDNA, both proteins and genes or cDNA Can be said to code the product of

  Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns.

  An “isolated nucleic acid” is a nucleic acid segment or fragment that is separated from sequences that naturally flank it, and usually refers to a DNA fragment that has been separated from, for example, sequences adjacent to the fragment, and is also naturally Refers to the sequence adjacent to a fragment in the genome present in The term is also used for nucleic acids that have been substantially purified from other components that are originally present with the nucleic acid, such as RNA, DNA or proteins that are originally present in the cell. Thus, for example, the term is a vector, a self-replicating plasmid or virus, or a separate molecule (cDNA, genomic or PCR or PCR or integrated into prokaryotic or eukaryotic genomic DNA, or independent of other sequences. Recombinant DNA existing as a cDNA fragment produced by restriction enzyme digestion). The term also includes recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence.

  In the context of the present invention, the following commonly used nucleobase abbreviations are used. “A” refers to adenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refers to thymidine, and “U” refers to uridine.

  A “vector” is a composition of matter that contains an isolated nucleic acid and can be used to supply the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides with ionic or amphiphilic compounds, plasmids, and viruses. That is, the term “vector” includes a self-replicating plasmid or virus. The term should also be construed to include non-plasmid and non-viral compounds that aid in the transfer of nucleic acid into cells, such as polylysine compounds and liposomes. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, and the like.

  “Expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence for expression. Expression vectors contain sufficient cis-acting elements for expression, and other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (eg, naked or contained in liposomes), viruses that incorporate recombinant polynucleotides, and the like.

(Description of the invention)
The present invention includes a method of promoting the proliferation of NSCs while maintaining their pluripotency. The method includes isolating NSCs using methods known in the art and co-culturing NSCs with BMSCs to promote their growth while maintaining NSC pluripotency. The cultivation of the two cell types is possible both in a contact-dependent method where NSC is in physical contact with BMSC and in a contact-independent method where NSC is not in physical contact with BMSC.

  The present invention relates to the discovery that the proliferative potential of NSCs (the ability of NSCs to replicate multiple times) can be increased by co-culturing with BMSCs. That is, examples of the present invention relate to the discovery that BMSCs can serve as support cells in co-culture systems for NSC growth. Based on the present disclosure, BMSC serves as a feeder cell layer for NSC and maintains NSC pluripotency, while at the same time supporting, but not limited to, growth factors, nutrients One skilled in the art will recognize that it is possible to supply factors including factors and cytokines. Also, the BMSC feeder cell layer can serve as a monolayer on which NSCs can grow.

  One skilled in the art can also recognize based on the present disclosure that BMSC and NSC can be co-cultured in the presence of other growth factors known in the art to promote NSC growth.

  In the present invention, BMSC and NSC can be co-cultured in the absence of exogenous LIF to maintain NSC pluripotency and at the same time promote NSC proliferation. The present disclosure shows that NSCs grow at a significantly higher level than that of NSCs cultured alone on plates coated in the presence of exogenous LIF (ie, plates coated with polyornithine / fibronectin). And show that it spreads. That is, the present invention provides a method of culturing NSCs without the need for coated plates and / or exogenous LIF.

  Further embodiments of the invention include methods for removing or separating BMSCs from NSCs in a BMSC and NSC co-culture. The present invention relates to the discovery that antibodies that bind to BMSC in co-culture can then be incubated and then removed from such co-culture by a separation step including, but not limited to, magnetic separation. One example of an antibody that binds to BMSC is an anti-CD13 antibody. The process of magnetic separation is achieved by using magnetic beads such as, but not limited to, Dynabeads (Dynal Biotech, Brown Deer, WI). In addition to the use of Dynabeads®, a MACS separation reagent (trademark, Miltenyl Biotec, Auburn, CA) can be used to remove BMSCs from co-cultures. As a result of the separation step, a purified NSC population can be obtained. FACS can also be used to remove BMSCs or alternatively to actively select NSCs.

  In the NSC culture / proliferation method of the present invention, NSC retains pluripotency (ability to differentiate into one of various cell types such as nerve cells, astrocytes, oligodendrocytes, etc.). In a further embodiment of the invention, NSCs grown using the methods of the invention are capable of differentiating more extensively (ie, at a greater rate) than growing or culturing NSCs using prior art methods. Is maintained.

  The NSC culture / proliferation methods presented herein solve the fundamental problems when using NSCs for the treatment of human diseases. That is, prior to being disclosed herein, it was difficult to isolate and propagate NSCs in culture (ie, it was difficult to induce NSCs to grow to a sufficient number). . The disclosure herein shows that NSCs can be grown and isolated in sufficient numbers for therapeutic use.

  The present invention also relates to the discovery that the expression of MHC molecules by NSC can be controlled by co-culturing NSC and BMSC. In addition to promoting NSC proliferation while maintaining pluripotency, the disclosure presented herein also co-cultured NSC and BMSC was also cultured using standard methods known in the art It shows that the upregulation and / or induction of MHC molecule expression by NSC is reduced compared to MHC molecule expression by NSC. That is, the present invention provides NSC culture methods that provide additional benefits over standard methods used to promote NSC growth in culture.

  The co-culture system provides an NSC culture method that provides significantly better effects than culturing NSCs on coated plates in the presence of NSCs alone or in the presence of exogenous LIF. As discussed elsewhere herein, the co-culture system provides the first method of producing large quantities of NSCs in a manner that does not require the use of coated plates or exogenous LIF. Furthermore, the co-culture system provides a method of providing additional effects and / or effects at a higher level compared to prior art methods of culturing only NSCs using standard NSC media. These benefits include, but are not limited to, maintaining NSC pluripotency and controlling NSC MHC molecule expression while simultaneously promoting NSC proliferation.

  In another example of the invention, NSCs can be co-cultured with BMSCs in the absence of exogenous LIF in order to reduce the upregulation and / or induction of MHC molecule expression by NSCs. That is, the present invention provides a method for culturing NSCs without the need to use exogenous LIF. The disclosure herein shows that in a co-culture system, BMSC can function as a feeder cell layer that supplies factors including, but not limited to, growth factors, trophic factors and cytokines to the co-cultured NSCs. Factors supplied by BMSC were co-cultured NSCs at a level greater than the benefit of culturing NSCs alone on coated plates in NSC medium in the presence of exogenous LIF for NSC growth and NSC expression of MHC molecules. It is thought to have a beneficial effect on

  The discovery that MHC molecule expression can be controlled using the methods disclosed herein provides a way to produce NSC populations useful for therapeutic, diagnostic, experimental applications, and the like. For example, when compared to methods known in the art, reduction of MHC molecule expression by NSCs using the methods disclosed herein provides a method of reducing NSC immunogenicity. Preferably, reducing the expression of MHC molecules by NSC provides a method for increasing the success of transplanting NSCs into a recipient.

  Co-culture of BMSC and NSC provides a method of controlling MHC molecule expression by NSCs in a contact-dependent or independent manner for the two cell types. In one embodiment of the invention, NSC can be co-cultured with BMSC in a contact-dependent manner. Without being limited by a particular theory, the physical interaction of BMSC and NSC triggers a beneficial effect for NSC proliferation and expansion. The physical interaction between the two cell types also contributes to the regulation of MHC molecule expression by NSCs. Preferably, contact-dependent co-culture of BMSC and NSC for two cell types is performed by MHC from another equivalent NSC cultured in the absence of BMSC on a coated plate using standard NSC medium supplemented with exogenous LIF. Reduces upregulation and / or induction of MHC molecule expression by NSCs as compared to molecular expression.

  In another example of the invention, NSCs can be co-cultured with BMSCs in a contact-dependent manner to regulate the expression of MHC molecules. The present invention finds that NSC and BMSC can be co-cultured in Coaster Transwell®, where a permeable membrane filter separates two cell types and prevents physical contact between NSC and BMSC. About. Permeable membrane filters allow factors secreted by BMSC to cross the membrane and consequently contribute to the NSC phenotype, for example, maintain NSC pluripotency, MSC molecules by NSC It can be used, for example, to promote the growth of NSC while regulating the expression of.

  As demonstrated by experiments with Transwell®, the present disclosure shows that there is no direct contact between the two cell types by means that BMSC supplements the medium with BMSC secreted factors in the medium. Shows that it can support the growth and expansion of NSCs in a co-culture system. Without being limited to a particular theory, factors secreted by BMSCs contribute to the NSC phenotype. That is, the present invention provides a method for co-culturing BMSC and NSC, in which BMSC does not need to be a feeder cell layer on which NSC directly grows. The present invention provides a contact-independent method of co-culturing BMSC and NSC in which NSCs benefit from BMSC in a co-culture system as a means of receiving BMSC secreted factors.

  Based on this disclosure, those skilled in the art will recognize that methods that prevent NSC and BMSC from being in physical contact with each other can be used for a co-culture system. For example, any system / device other than Transwell® can be used to obtain contact-independent co-cultured cells. Such systems / devices include, but are not limited to, filters and membranes with pore sizes that prevent direct contact between the two cell types but allow passage of the filter / membrane factors. An advantage of using such a system / device to co-culture BMSCs and NSCs is to allow a method of culturing NSCs in a system that has a continuous source of factors for NSC growth. Therefore, the present invention comprises an arrangement comprising an isolated NSC, an isolated BMSC, an NSC growth medium and a neural stem cell culture apparatus comprising a factor secreted by the isolated BMSC, and means for preventing NSC and BMSC from contacting each other. Includes.

  Based on the discovery that BMSC can support the growth of NSC in a contact-independent co-culture system through supplying factors secreted from BMSC into the medium, a medium conditioned by BMSC is It was assessed whether it could support NSC proliferation in the form of maintaining pluripotency while simultaneously promoting NSC proliferation and regulating the expression of MHC molecules by NSC. The present disclosure demonstrates that media conditioned by BMSC can support NSC growth and have significantly beneficial if not more efficient than contact-dependent. That is, the present invention provides a method of using bone marrow stromal cell conditioned medium (BMSC-CM) for culturing NSCs without using a co-culture system. The use of BMSC-CM to culture NSCs also provides another way to produce NSC populations that have properties equivalent to those cultured using a co-culture system.

  Furthermore, the present disclosure demonstrates that BMSC-CM can be an alternative to the use of NSC media supplemented with exogenous LIF to culture NSCs. The present disclosure demonstrates that the number of cells produced after culturing NSCs with BMSC-CM is comparable to the number of cells produced using NSC medium supplemented with exogenous LIF. That is, the present invention provides a method of using BMSC-CM as a useful factor source for inducing NSC cell proliferation at a rate equal to or higher than when using NSC medium supplemented with exogenous LIF.

  It has also been demonstrated that BMSC-CM can be an alternative to using coated plates such as polyornithine / fibronectin coated plates for NSC culture and growth using NSC media. The disclosure herein demonstrates that NSCs can be grown using BMSC-CM on uncoated plates. Even if exogenous LIF is present in the NSC medium on the coated plate, the growth of NSC cultivated there alone and NSC using BMSC-CM on the uncoated plate is at least equivalent. It has been observed that, or beyond. That is, the present invention includes a method of culturing NSCs using BMSC-CM without the need for coated plates and exogenous LIF.

  Also, the use of BMSC-CM provides a method for culturing NSCs in an effective manner for NSCs without using BMSCs in a co-culture system. The present disclosure demonstrates that BMSC-CM supports NSC culture and provides NSCs with an effect equivalent to that seen when using a co-culture system comprising BMSC and NSC. As such, the present invention includes a method of culturing NSCs using BMSC-CM to maintain NSC pluripotency, regulate the expression of MHC molecules and at the same time promote NSC proliferation.

  As demonstrated herein, BMSC is used to make bone marrow stromal cell conditioned medium (BMSC-CM). BMSC-CM is a medium conditioned by BMSC during culture by culturing BMSC in NSC medium and in particular secreting growth factors, trophic factors and / or cytokines into NSC medium. BMSC-CM is a growth factor, trophic factor, and / or cytokine secreted from BMSC, and includes but is not limited to LIF, brain-derived neurotrophic factor (BDNF), epidermal growth factor receptor (EGF), base Fibroblast growth factor (bFGF), FGF-6, glial cell-derived neurotrophic factor (GDNF), granulocyte colony stimulating factor (GCSF), hepatocyte growth factor (HGF), IFN-γ, insulin-like growth factor binding Protein (IGFBP-2), IGFBP-6, IL-lra, IL-6, IL-8, monocyte chemotactic protein (MCP-1), mononuclear phagocyte colony stimulating factor (M-CSF), nerve Nutritional factor (NT3), tissue metalloprotease inhibitor (TIMP-1), TIMP-2, tumor necrosis factor (TNF-β), vascular endothelial growth factor (VEGF), VEGF-D, urokinase plasminogen activation Factor receptor (uPAR), bone morphogenetic protein 4 (BMP4), IL1-a, IL-3, leptin, stem cell factor (SCF), stromal cell-derived factor-1 (SDF-1), platelet-derived growth factor-BB ( PD GFBB), tumor cell growth factor beta (TGFβ-1), and TGFβ-3.

  BMSC-CM is useful for proliferating and expanding NSC while maintaining NSC's multipotency. The use of BMSC-CM provides a way for NSCs to grow and expand, providing NSCs with growth factors, trophic factors, and / or cytokines that are specifically secreted from BMSCs. The use of BMSC-CM provides a method of inducing NSC growth on uncoated surfaces at a rate comparable to or greater than when cultured with NSC medium supplemented with exogenous LIF. That is, the present invention provides compositions and methods using BMSC-CM to produce large amounts of therapeutic NSCs.

  In addition to using BMSC-CM to support NSC proliferation while maintaining pluripotency, culturing NSCs in BMSC-CM regulates the expression of MHC molecules by NSC It also demonstrates that it provides a way to do this. Preferably, culturing NSCs in BMSC-CM reduces the expression of MHC molecules by NSCs compared to the expression of MHC molecules by other equivalent NSCs cultured in NSC medium in the presence of exogenous LIF. . That is, culturing NSCs in BMSC-CM reduces and / or suppresses MHC molecule expression upregulation.

  The use of BMSC-CM to regulate the expression of MHC molecules by NSCs means that NSCs grown in the presence of BMSC-CM express MHC class II molecules under the conditions used for detection of MHC class II molecules And based on the discovery that it showed lower MHC class I expression levels compared to other equivalent NSCs cultured in NSC medium with exogenous LIF. This finding is consistent with the finding that expression of MHC molecules by NSC is reduced after co-culture of NSC and BMSC in either contact-dependent or contact-independent manner. In any case, the reduction of MHC molecule expression by NSCs using either NSC cultures in BMSCs or BMSC-CM as a feeder cell layer in a contact-dependent or contact-independent manner is caused by NSCs. A method of reducing molecular expression is provided. The discovery that the expression of MSC molecules can be modulated using the methods disclosed herein provides a way to produce therapeutically useful NSC populations. In addition, reduction of MSC molecule expression by NSCs using the methods disclosed herein also provides a way to increase the probability of successful transplantation into a recipient.

  Based on this disclosure, the present invention encompasses the use of BMSC-CM to cultivate and proliferate NSC while simultaneously reducing MHC molecule expression by NSC. That is, the present invention shows that the use of BMSC-CM for NSC growth is more up-regulated on MSC molecules on NSC compared to other equivalent NSCs cultured in NSC medium in the presence of exogenous LIF. It is based on the discovery that it is reduced and / or suppressed. As such, the present invention includes a method of producing cells useful for therapy.

  It is widely established that genetically different individuals (allogeneic) cell transplantation always carries the risk of transplant rejection. Almost all cells express the product of MHC class I molecules, which are the major compatible antigens. In addition, many cell types are induced to express MHC class II molecules when exposed to inflammatory cytokines. Allograft rejection is primarily mediated by T cells having CD4 and CD8 subclasses that recognize MHC class I and class II molecules. The main goal in transplantation is the permanent engraftment of donor grafts without inducing the graft rejection immune response that occurs in the recipient. As such, the present invention provides a method for cultivating NSCs prior to transplantation using the methods for reducing MHC molecule expression by NSCs disclosed herein to provide a recipient side for transplanted NSCs in a recipient. Methods for reducing and / or eliminating the immune response by the cells. Without being limited to a particular theory, reduced expression of MHC molecules by NSCs using the methods disclosed herein reduces the amount of MHC molecules on the NSC cell membrane, and as a result, in recipients. Reduces the immunogenicity of NSCs.

  The present invention can also be used to obtain NSCs that express exogenous genes, such that, for example, NSCs can be used for cell therapy or gene therapy. That is, the present invention enables the production of large amounts of NSCs that express exogenous genes. An exogenous gene is, for example, an exogenous version of an endogenous gene (ie, a wild type version of the gene can be used to replace a defective allele containing a mutation). The exogenous gene can include a trophic factor for central nervous system regeneration and a cytotoxic gene targeting cancer. Generally, although not necessarily, the exogenous gene is covalently linked (ie, fused) to one or more additional genes. As an example of an “additional gene”, a gene used for “positive” selection that selects a cell into which an exogenous gene has been incorporated and an exogenous gene have been incorporated into the same chromosomal site as the endogenous gene. Examples include genes used for “negative” selection to select cells, or both.

  NSCs obtained by the method of the present invention are differentiated into cells such as neurons, astrocytes and oligodendrocytes by selection of culture conditions known in the art for differentiating NSCs into selective cells. It is possible to guide.

  As described in this disclosure, cultured and expanded NSCs can be used to treat various diseases known in the art as being capable of treatment with NSCs before or after differentiation into a selected cell type. Available to: NSCs are useful in treatments that may include treatments with and without exogenous genes inserted into the NSC. Examples of such diseases include, but are not limited to, brain trauma, Huntington's disease, Alzheimer's disease, Parkinson's disease, spinal cord injury, stroke, multiple sclerosis, cancer, central nervous system lysosomal disease, head trauma Is included.

(Isolation of NSC and BMSC)
NSCs are obtained from the central nervous system of mammals, preferably humans. These cells can be obtained from a variety of tissues including, but not limited to, the forebrain, hindbrain, whole brain, and spinal cord. NSCs are isolated using methods detailed elsewhere herein, or known methods, such as those disclosed in US Pat. No. 5,958,767, which is incorporated herein by reference in its entirety, Can be cultured. Other methods of isolating NSC are known and can be readily used by those skilled in the art, including methods that will be developed in the future. The present invention is in no way limited to these or other methods for obtaining the desired cells.

  NSCs can be isolated from many different types of tissues, eg, individual cells from donor tissues, from the extracellular matrix of connective tissue, or from commercially available NSC sources. In one example, brain tissue is removed by aseptic manipulation and methods including enzyme treatments such as trypsin and collagenase, or methods using physical separation means such as grinding or treatment with blunt instruments, etc. Cells can be separated using any known method. Separation of neural cells and other multipotent stem cells can be performed in a sterile tissue culture medium. The separated cells are centrifuged at a low speed of 200-2000 rpm, usually 400-800 rpm, the suspension medium is aspirated, and the cells are resuspended in the medium.

  BMSC raw materials and methods for obtaining BMSCs from these raw materials are known. Virtually BMSCs can be obtained from any bone marrow, such as bone marrow obtained by aspiration of the iliac crest of a human donor. Methods for obtaining bone marrow from a donor are well known in the art and are described, for example, in US Pat. No. 6,653,134 and International Publication WO 96/30031, which are incorporated herein in their entirety. Human mesenchymal stem cells can also be purchased from Cambridge (Walkersville, MD)

(Use of isolated neural stem cells)
Isolated neural stem cells can be used in various forms. These cells can be used to reconstruct mammalian cells that are lost due to illness or injury. By genetic recombination of autologous or allogeneic neural stem cells, genetic diseases can be treated to correct genetic defects or prevent disease. Diseases associated with a deficiency of certain secretions such as hormones, enzymes, growth factors, etc. can also be treated using NSCs. Central nervous system disorders can result in a number of pains such as neurodegenerative diseases (e.g. those of Alzheimer's or Parkinson's disease), severe brain damage (e.g. stroke, head trauma, cerebral palsy, tumor resection, chemotherapy and radiation therapy). Law), as well as numerous central nervous system dysfunctions (eg, depression, epilepsy, and schizophrenia). Diseases including but not limited to Alzheimer's disease, multiple sclerosis (MS), Huntington's chorea, amyotrophic lateral sclerosis (ALS), and Parkinson's disease are all specific locations in the central nervous system. Associated with the degeneration of a neuron in that cell, or area of the brain, will not be able to perform its original function. The isolated and cultured NSCs described herein can be used as progenitor cell material and against constrained cells to treat these diseases. NSCs can be used as a source of trophic factors that stimulate the regeneration of endogenous stem cells and the central nervous system.

  The cultured NSCs described herein can be frozen at liquid nitrogen temperature, stored for a long time, thawed and used again. Usually, cells are stored in NSC medium of DMSO 10%, NSC 90%. Once thawed, the cells can be grown using methods detailed elsewhere herein.

(gene recombination)
In order to produce molecules such as trophic factors, growth factors, cytokines, and neurotrophic factors useful for NSC culture, the gene of the cell of the present invention is recombined by introducing exogenous genetic material into the cell. be able to. For example, recombine BMSC genes to express and secrete epidermal growth factor (EGF) at a higher level compared to BMSCs that have not been genetically modified to express factors such as EGF be able to. Without being limited to any particular theory, a BMSC that has been genetically modified to express and secrete EGF is compared to another equivalent BMSC that has not been genetically modified to express such factors. Expression and secretion at increased concentrations.

  An advantage of using the recombinant BMSC in the co-culture system is that the exogenous factor can be continuously supplied to the recombinant BMSC. Exogenous factors serve to benefit cultured BMSCs and / or NSCs. In addition, exogenous genetic material introduced into BMSC can contribute to the secretion of other endogenous factors from recombinant BMSC. Furthermore, genetically modified BMSCs can contribute to endogenous factor secretion from neighboring cells. That is, the present invention encompasses the use of genetically modified BMSCs to provide a continuous supply of exogenous factors to a co-culture system, and sometimes exogenous genetic material introduced into BMSCs is gene Contributes to the secretion of endogenous factors from recombinant BMSCs and / or neighboring cells. In any case, exogenous factors and / or endogenous factors secreted from BMSCs provide beneficial factors for NSC culture and growth.

  Furthermore, in order to produce BMSC-CM with increased EGF concentration, genetically modified BMSC can be used to express and secrete factors such as EGF. In addition to bringing increased concentrations of exogenous factors to BMSC-CM, genetically modified BMSCs can also contribute to secretion of endogenous factors from genetically modified BMSCs and / or neighboring cells . The factors include, but are not limited to, LIF, brain-derived neurotrophic factor (BDNF), epidermal growth factor receptor (EGF), basic fibroblast growth factor (bFGF), FGF-6, glial cell-derived nerve Nutritional factor (GDNF), granulocyte colony stimulating factor (GCSF), hepatocyte growth factor (HGF), IFN-γ, insulin-like growth factor binding protein (IGFBP-2), IGFBP-6, IL-lra, IL-6 , IL-8, monocyte chemotactic protein (MCP-1), mononuclear phagocyte colony stimulating factor (M-CSF), neurotrophic factor (NT3), tissue metalloprotease inhibitor (TIMP-1), TIMP-2, tumor necrosis factor (TNF-β), vascular endothelial growth factor (VEGF), VEGF-D, urokinase plasminogen activator receptor (uPAR), bone morphogenetic protein 4 (BMP4), IL1-a IL-3, leptin, stem cell factor (SCF), stromal cell derived factor-1 (SDF-1), platelet derived growth factor-BB (PDGFBB), tumor cell growth factor beta (TGFβ-1), and TGFβ-3 including.

  The benefit of using genetically modified BMSC for BMSC-CM production is that when compared to BMSC-CM produced from another equivalent BMSC that has not been genetically modified, for example Is to increase the concentration of exogenous factors such as EGF. Due to the increased concentration of EGF secreted from the genetically modified cells, more EGF is present in BMSC-CM. Furthermore, an increase in the concentration of EGF secreted from the recombinant BMSC can contribute to the secretion of the recombinant BMSC and / or other endogenous factors from neighboring cells. BMSC-CM with increased concentrations of EGF and / or other factors can be useful for culturing and proliferating NSCs.

  In another aspect, the HSV-thymidine kinase or green fluorescent protein (green fluorescent protein) used for genetic modification of BMSC and subsequent NSC growth in BMSC co-culture followed by NSC removal by treatment with ganciclovir or separation with a flow cytometer ( A gene such as GFP) can be expressed.

  In addition to recombining BMSC genes, the present invention also includes recombinant NSCs. Genetically modified NSCs can be used to replace defective cells in an individual. The present invention can also be used to express a desired secreted protein. In other words, the NSC can be isolated, introduced with the gene for the desired protein, and can be introduced into an individual where the desired protein is produced and acts, or otherwise produces a therapeutic effect. This aspect of the invention relates to gene therapy in which a therapeutic protein is administered to an individual by means of introducing a recombinant NSC into the individual. Genetically modified NSCs are cultured, isolated using the methods disclosed herein, and introduced into an individual that benefits from the protein being expressed and secreted by NSCs in the body.

  According to the present invention, a genetic structure comprising a nucleotide sequence encoding a heterologous protein is introduced into the NSC. That is, the cells are modified to introduce genes whose expression normally has a therapeutic effect on the individual. According to some aspects of the invention, the NSC from a patient, another patient, or a non-human animal replaces the defective gene and / or introduces a gene whose expression has a therapeutic effect on the individual Can be modified.

  In any case where a genetic construct is introduced into a cell, the heterologous gene is operably linked to regulatory sequences necessary to achieve expression of the gene in the cell. Such control sequences include promoters and polyadenylation signals.

  Preferably, the genetic construct is provided as an expression vector that is operably linked to regulatory sequences essential for the coding sequence to be expressed by the cell when the vector is introduced into the cell. Contains the coding sequence for the protein. The coding sequence is operably linked to regulatory sequences necessary for expression of the sequence in the cell. The nucleotide sequence encoding the protein may be cDNA, genomic DNA, synthetic DNA, or a hybrid thereof, or an RNA molecule such as mRNA.

  A genetic construct contains a nucleotide sequence that encodes a beneficial protein that operably binds to a regulatory element and exists in the cell as a cytoplasmic functional molecule, a functional episomal molecule, or in association with the chromosomal DNA of the cell . The exogenous genetic material may be introduced into the cell and exist as a separate genetic material in the form of a plasmid. Alternatively, linear DNA that can bind to the chromosome can be introduced into the cell. When introducing DNA into a cell, a reagent that promotes binding of the DNA to the chromosome can be added. Further, a DNA sequence useful for promoting binding may be contained in the DNA molecule. As another method, RNA may be introduced into cells.

  Control elements for gene expression include a promoter, a start codon, a stop codon, and a polyadenylation signal. It is preferred that these elements function in the cells of the invention. Furthermore, it is preferred that these elements be operably linked to the nucleotide sequence encoding the protein so that the protein can be produced because the nucleotide sequence is expressed in the cell. In general, start and stop codons are considered to be part of a nucleotide sequence that encodes a protein. However, it is preferred that these elements are functional in the cell. Similarly, the promoter and polyadenylation signal must be functional in the cells of the invention. Examples of promoters useful in the practice of the present invention are promoters active in many cells such as, but not limited to, cytomegalovirus promoter, SV40 promoter, and retrovirus. Another example of a promoter useful in the practice of the present invention includes, but is not limited to, tissue specific promoters, ie, promoters that function in some tissues but not others, and specific or general Includes promoters of genes that are normally expressed in cells with or without enhancer sequences. In some embodiments, a promoter is used that constitutively expresses the gene in the cell with or without an enhancer sequence. Enhancer sequences are provided in such examples where appropriate or necessary.

  The cells of the invention can be transfected using known techniques that are readily available to those with ordinary knowledge in the art. Exogenous genes can be introduced into cells by standard methods used to introduce genetic constructs into cells that express the protein encoded by the gene. In some examples, the cells are calcium phosphate precipitated transfection, DEAE dextran transfection, electroporation, microinjection, liposome-mediated transfer, chemical-mediated transfer, ligand-mediated transfer, or recombinant. Transfected by type viral vector transfer.

In some embodiments, recombinant adenoviral vectors are used to introduce DNA into cells along with the desired sequence. In some embodiments, recombinant retroviral vectors are used to introduce DNA into cells along with the desired sequence. In some embodiments, standard CAP0 _4, DEAE dextran, or transfection techniques through the lipid carrier, used to capture the differentiated cells of the desired DNA. Standard antibiotic resistance selection techniques can be used to identify and select transfected cells. In some embodiments, DNA is introduced directly into cells by microinjection. Similarly, known electroporation or particle bombardment techniques can be used to introduce foreign DNA into cells. Usually, the second gene is co-introduced or linked to a therapeutic gene. In general, the second gene is a selectable antibiotic resistance gene. Transfected cells can be selected by culturing in antibiotics that kill cells that do not take up a selectable gene. In many cases where the two genes are not joined and are co-introduced, the cell that survives antibiotic treatment has both genes in it and expresses both of them.

  The following examples are presented in order to more fully illustrate the preferred embodiments of the invention. These examples in no way limit the scope of the invention as defined by the appended claims.

(BMSC as support cells for NSC growth and expansion)
NSC culture is difficult because NSC culture requires growth factors and a specific substratum to promote growth and expansion. With respect to growth factors, the addition of exogenous LIF to defined growth media containing FGF and / or EGF and no serum significantly promotes NSC growth. As stated elsewhere herein, the combination of exogenous LIF and culture dish coating further increases the level of NSC proliferation while maintaining pluripotency.

  Bone marrow stromal cells (BMSCs) can be easily obtained and a substantially identical cell population can be grown in culture. In addition, BMSCs secrete some trophic factors that will promote NSC growth. Thus, this example demonstrates that BMSC can play a role in supporting cells in a co-culture system for NSC growth.

  The materials and methods used in the experiments shown in this example are now described.

(Establishment, maintenance and characterization of human fetal neural stem cells (NSC))
Human brains (11-14 week fetuses) were obtained from Advanced Bioscience Resources Inc. (Alameda, Calif.). Brain tissue was ground in cold phosphate buffered saline (PBS). Cells are pelleted by centrifugation and 10 ml of NSC growth medium (DMEM / F12, 8 mM glucose, glutamine, 20 mM sodium bicarbonate, 15 mM HEPES, 8 μg / ml heparin, N ₂ supplement, lOng / ml bFGF, 20 ng / ml EGF ). Cells were seeded onto T-25 cm flasks coated with polyornithine and cultured at 37 ° C. in a 5% CO ₂ incubator. Cultures were fed with 50% of the medium being replaced with fresh medium every other day and passaged with trypsinization every 14 days. Cells were cryopreserved in 10% DMSO NSC medium in a liquid nitrogen gas phase. Cells were thawed and first subcultured for about 1-2 in the presence of bFGF and EGF and then seeded in complete growth medium supplemented with 10 ng / ml LIF.

  Alternative NSC growth media can be used for the methods disclosed herein. For example, cells can be cultured in Neurobasal ™ medium (Invitrogen, Carlsbad, Calif.) Supplemented with L-glutamine, bFGF, EGF, and B27 and free of retinoic acid. Another NSC growth medium is NeuroCult® (Stem Cell Technologies, Vancouver BC, Canada) supplemented with growth factors. Without being limited to a particular theory, any medium can be used for NSC culture. A suitable medium, however, allows the cells to grow and expand while maintaining the cell's potential to differentiate into multiple cell types.

  For characterization, NSCs were seeded on coated chamber slides at different passages, fixed with 4% paraformaldehyde, and stained for nestin and glial fibrillary acidic protein (GFAP). . NSCs were differentiated for approximately 14 days by treating NSCs with Neurobasal ™ medium, B27 supplement, and BDNF while removing bFGF, EGF, and LIF. Different differentiation conditions may be used to differentiate the cells into more specific lineages. Cells were fixed and stained for microtubule-associated protein (MAP2; neuronal marker) and GFAP (astrocytic marker). To identify neuronal subtypes, cells were stained with anti-γ-aminobutyric acid (GABA), anti-tyrosine hydroxylase (TH). The primary antibodies used were human-specific nestin 10-fold diluted (R & D Systems), MAP2 500-fold diluted (Sigma), GFAP 1000-fold diluted (Dako), O4 100-fold diluted, NG-2 200 Double dilution (Chemicon). The secondary antibodies used in these experiments were diluted with the trademark Alexa Fluor 488 avian anti-mouse 500-fold (Molecular Probes) and the trademark Alexa Fluor 594 avian anti-rabbit 500-fold (Molecular Probes). is there.

  NSC is a flow cytometer for expression of several markers including hematopoietic cell markers CD45 and CD14, stem cell markers CD34, CD133 and CD56, and immunogenic / stimulatory markers CD80, CD86, MHC class I and MHC class II. Was analyzed.

  Quantitative analysis of nestin-expressing cells was also measured using flow cytometry. This analysis was performed using anti-nestin (R & D) and goat anti-mouse Ig conjugated with Alexa-fluor488 ™. NSCs were fixed and permeabilized by using Becton-Dickinson's Cytofix / Cytopalm Kit (trademark) with slightly different instructions.

(Establishment, maintenance and characterization of human bone marrow stromal cells (BMSCs))
BMSC was produced by methods known in the art. For example, human bone marrow was collected by needle aspiration. Nucleated cells were counted in the bone marrow. Bone marrow was diluted with PBS and mixed with Hesspan®. The Hesspan / cell suspension mixture was allowed to stand for about 45 minutes to 1 hour. Meanwhile, red blood cells (RBC) settled, and nucleated cells gathered in the supernatant layer. After removal of RBCs, nucleated cells were washed and counted.

  Nucleated cells were cultured in a DMEM low glucose medium containing 10% fetal calf serum, for example, in a tissue culture processing vessel made of polystyrene. The first culture was observed continuously for about 12 to 17 days with medium changes every 3 to 4 days. When the number of adherent spindle cells was sufficient, the culture was passaged by removing the adherent cells using trypsin. The cells were transferred to a separate container, and continued to be cultured for about 1 week while changing the medium once every passage.

  The cells were tested for unification using specific markers by flow cytometry. BMSCs at passage 1 or 2 (PI or P2) that were CD45 negative and over 90% CD90 and CD13 positive were used for co-culture experiments.

(BMSC culture in NSC growth medium)
BMSCs were seeded at different densities on 6-well plates and allowed to adhere overnight in BMSC media (low glucose, DMEM with 10% fetal bovine serum tested lot). The next day, the medium of one plate was replaced with NSC medium (DMEM-F12, N2-supplement, EGF, 20 ng / ml, bFGF, 10 ng / ml, 8 llg / ml heparin, P / S). The remaining plates were filled with fresh BMSC medium. Cells were given fresh BMSC or NSC medium every 3 days. One set of cultures was treated with trypsin after 7 days and the number of cells counted. Another set of cells was collected after 12 days and counted. As shown in Table 1 and FIG. 1, when cultured in BMSC medium, BMSC proliferated much more than when cultured in NSC medium. Compared to what was observed in BMSC medium, the initial increase in cell number that cells showed in NSC medium was low. However, after 7 days, there was almost no increase in the number of BMSC cells in NSC medium. Cells continued to grow for over 12 days in BMSC medium, and eventually showed high confluence. In NSC medium, BMSCs did not form confluent cells, but were morphologically normal except for visible cell death. In BMSC medium, a contrasting morphology of the cells was observed.

  These results indicate that when NSC and BMSC are cultured together in NSC medium, BMSC does not compete with NSC and grow more than NSC.

(Co-culture of BMSC and NSC: survival and proliferation of NSC on BMSC compared to survival and proliferation of NSC on coated plate)
Human BMSC (hBM-03-016, PI) was thawed, washed, counted, and seeded with 7500 cells per well in two 12-well plates. At the same time, another 12-well plate was coated with 15 μg / ml polyornithine followed by 10 μg / ml human fibronectin and left overnight. On the next day, hNSC in culture was collected and resuspended in NSC medium containing EGF and FGF. The NSC used was hHB-007, P7, which was cultured in an NSC medium containing EGF, FGF and LIF. Most of these cells proliferate as neurospheres. When these cells were seeded in BMSCs or coated holes, the neurospheres were split to be as single as possible. Three sets of NSCs were seeded on BMSCs or coated plates under the following conditions.
1. 25,000NSC + NSC medium without LIF
2. 25,000NSC + NSC medium, + LIF
3. 50,000NSC + NSC medium, no LIF
4.50,000NSC + NSC medium, + LIF

  The culture was fed every other day by replacing 50% of the spent medium with fresh medium. Phase contrast micrographs of the cells were taken from time to time. After 12-13 days in culture, cells were harvested using trypsin. Briefly, cells were placed with 0.05% trypsin for 5 minutes or until all cells were lifted from the dish. Subsequently, trypsin was neutralized by soybean trypsin inhibitor (SBTI). The cells were washed with phosphate buffered saline (PBS) and then counted on a hemocytometer using trypan blue staining solution. Two different sized cells were seen under the microscope, large BMSC and small NSC. The two groups were counted separately. Initial counts were measured on day 12 by storing two of the three homotypic cultures.

  NSCs were seeded on BMSC, adhered, and extended as a single layer in the high concentration region. It was not difficult to change the medium on these cells. On the other hand, especially NSCs (FIG. 2) grown in the presence of exogenous LIF as neurospheres did not adhere well, and extreme care had to be taken when changing the medium. Occasionally, centrifugation of the aspirated media was required to prevent neurosphere loss. NSCs did not survive unless seeded with BMSC when seeded at low density. NSCs grown during co-culture formed a morphologically small oval cell network on the widely spread planar BSCs. NSCs that formed isolated colonies were sometimes surrounded by a ring of fibroblast BMSCs (FIG. 3). During culturing, the number of NSCs increases and the NSC colonies grow as each colony grows. NSCs survive and grow alone only in the presence of exogenous LIF and at high seeding densities. However, in the case of co-culture, significant expansion of NSC occurs even in the absence of exogenous LIF. In other words, it seems that BMSC served as a feeder cell layer and also provided nutritional support to NSC. A significant expansion of NSCs was obtained in the co-culture, which corresponded to the expansion of NSCs grown in exogenous LIF in coated (ie, polyornithine / fibronectin coated) dishes. Table 2 represents the number of NSCs and BMSCs collected after 12 days of culture.

(Characterization of NSCs cultured on BMSC: Nestin expression)
To evaluate NSC nestin expression in co-culture with BMSC by immunostaining, culture was performed on coverslips. Cover slips (lOmm) were placed in 24-well dishes coated with polyornithine followed by human fibronectin as described elsewhere herein. One set of coverslips was seeded with BMSC (35,000 / coverslips) and the cells were allowed to adhere overnight. The next day, NSCs grown in culture (THD-015 + LIF, P12) were collected and counted. 25000 NSC / hole were seeded on BMSCs or directly coated coverslips. Some of the holes with BMSC were cultured alone without NSC. All different cultures were performed in either NSC medium or NSC medium + LIF. Cells were given 50% fresh media every other day for 10 days. After 10 days, some cultures were fixed and prepared for nestin staining. Other cultures were differentiated for 2 weeks.

  To fix the culture, the culture was washed once with PBS. Then 4% paraformaldehyde was added, left at room temperature for 15 minutes, and washed 3 times with PBS. Cultures fixed at this point were stored in PBS at 4 ° C. or stained immediately.

  The nestin staining after the culture was completed by staining with anti-nestin antibody + anti-GFAP antibody. The culture includes BMSC + NSC, BMSC + NSC + LIF, BMSC alone, BMSC + LIF, NSC alone, and NSC + LIF. One sample in duplicate was used as a control (no primary antibody).

  NSCs cultured alone in NSC medium expressed nestin regardless of the presence or absence of exogenous LIF. When cultured in NSC medium + LIF, in addition to nestin expression, NSC simultaneously expressed GFAP.

  All co-cultures showed numerous nestin-positive cells spreading on the surface of BMSC (FIGS. 4A-4F). BMSC itself showed very light background staining (FIGS. 4G-4H). The morphology of nestin positive cells was heterogeneous. Many nestin positive cells were also positive for GFAP, while other cells were nestin positive and GFAP negative. Some nestin positive cells were small, round or oval with or without one or two filamentous stretches. Some, nestin-GFAP double positive cells were large, flat, and similar to astrocytes. At first glance, there was no apparent difference in the presence or absence of exogenous LIF when the co-culture was cultured.

(Differentiation of co-culture)
After 10 days of culture in NSC medium, the co-culture was subjected to a 2-week differentiation plan. The differentiation plan included two supplies for each of 1) DMEM / F12 and N2 supplement, no EGF or FGF, 2) DMEM / F12 + B27 supplement, and 3) DMEM / F12 + B27 + BDNF. Differentiation cultures were fixed with paraformaldehyde and then stained with the following antibody combinations: for example, nestin / GFAP, MAP2 / GFAP and O4 / GFAP. In all cases, GFAP was detected with red fluorescence (Alexa594 ™), while nestin, MAP2 or O4 was detected with green fluorescence (Alexa488 ™). Cultures were counterstained with DAPI and all nuclei were labeled with blue fluorescence.

  NSCs proliferate while retaining the ability to differentiate into neurons and astrocytes on BMSCs (FIGS. 5A-5D). After growing in differentiation medium, some cells express the neuronal marker MAP-2. It is a small cell with an elliptical nucleus and a bipolar or multipolar cell body. The process is extensive and a complex network of neurons is formed on the surface of BMSCs or cells stained with GFAP. GFAP positive astrocytes are also present throughout the culture. Compared to neurons, they are large, polygonal, and flat. Extensive networks of neurons form a three-dimensional network of cells, which cannot be measured exactly, but in general, there are neurons that are equal to or more than astrocytes. In the diluted solution used, staining with O4 antibody revealed that no cells differentiated into oligodendrocytes. It seems that a longer differentiation period is required to produce oligodendrocytes. However, by using NG2 (chondroitin sulfate proteoglycan found on the surface of oligodendrocyte precursors) as a marker of oligodendrocyte differentiation, the presence of oligodendrocyte cells is evaluated, and there are almost no NG2 positive cells It was observed. Nestin staining of differentiated co-cultures revealed some nestin-positive, some GFAP-positive astrocytes, which show morphological differences from the small filamentous cells seen before differentiation (FIG. 6). The presence or absence of exogenous LIF during culture did not differ in NSC differentiation in co-culture. It was observed that when NSCs cultured alone differentiated as described above, they became neurons (MAP2 positive) and astrocytes (GFAP positive) (FIG. 7).

  When subjected to similar differentiation conditions with BMSC alone, they showed a faint and diffuse staining of nestin and GFAP, and GFAP was somewhat stronger in cells cultured with exogenous LIF added. The cell morphology remained similar to BMSC and was not astrocytes. Rare cells in culture were seen where high nestin or MAP2 positivity suggests some differentiation in NSC or neuronal cell lines (FIG. 8).

(Depletion of BMSC from BMSC and NSC co-culture)
Cells from BMSC and NSC co-cultures can be isolated by incubating the co-cultures with mouse anti-human CD13 antibodies (BMSC positive and NSC negative). BMSCs can be separated from NSCs by using magnetic beads bound to pan mouse IgG. Non-binding cells that are NSCs are analyzed by a fluorescence activated cell sorter (FACS) or returned to culture. In FACS analysis, different BMSC marker antibodies (mouse anti-human CD105) are used to detect contaminating BMSCs at the same time NSC is detected using mouse anti-human CD133.

  BMSCs are seeded in 6-well dishes at 150,000 cells / well and allowed to attach overnight in BMSC medium. NSC, THD-hWB-015 + LIFP17 growing in culture was harvested and resuspended to break the neurospheres. BMSC medium was removed from the BMSC culture and the recovered NSCs were seeded on the surface of BMSC in NSC medium with or without exogenous LIF, including EGF and FGF (75,000 cells / well). The co-culture was maintained in BMSC medium for 13 days. Cells were replenished by replacing half of the spent medium with fresh medium every other day or over the weekend.

  On day 13, co-cultures were harvested using trypsin followed by soybean trypsin inhibitor. The cell mixture was resuspended in PBS containing 0.1% BSA. The cell mixture was incubated with anti-CD13 antibody for 30 minutes on ice. Cells were then washed with PBS / 0.1% BSA by centrifugation. At the same time, Dynalbead ™ -pan mouse IgG was washed 3 times with PBS / 0.1% BSA each time it was separated on a magnetic particle concentrator (Dynal-MPC). Washed cells were incubated for 30 minutes with washed magnetic beads in PBS / 0.1% BSA at 4 ° C. on a tilt / rotator (Dynal Sample Mixer ™). The tube with the cell-bead mixture was placed in the MPC for 2-3 minutes. Cells attached to the beads adhere to the side of the test tube closest to the magnet. The supernatant was collected and placed in a separation test tube.

  Some of the cells in the supernatant were used for FACS analysis, and some of the cells were seeded on chamber slides coated with polyornithine and fibronectin. FACS analysis was performed using anti-CD105 (recognizes BMSC) and anti-CD133 (recognizes NSC). Cells seeded on chamber slides were cultured in NSC medium for 1-2 days, fixed and immunostained with nestin / GFAP staining.

  After 2 weeks, NSCs grew on the BMSC layer. Several large colonies were seen early on, presumably originating from small neurospheres. Other NSCs that emerged as one or several cells grew into several small colonies of NSCs after 2 weeks.

  After BMSC magnetic disappearance, the separated NSCs were analyzed by FACS. More than 90% of the cells were CD133 positive (FIG. 9B). Less than 2% were CD105 positive (FIG. 9A). Cells seeded on chamber slides attached to the dish and were morphologically similar to NSCs. Nestin cell staining was used to confirm the presence of NSCs. The BMSC-bead mixture placed in the culture was morphologically similar to BMSC and attached to the tissue culture flask with a large number of bound magnetic beads. A simple culture with an antibody that binds to BMSC, followed by separation with a magnet, shows that the enriched NSC population remains, indicating that the BMSC is well depleted from the co-culture.

(Co-culture scale-up and NSC growth promotion using minimal BMSC as support cell layer)
In this experiment, co-culture was expanded to T-75 tissue culture flasks. BMSCs (donors BM-022, PI and BM-024, PI) were seeded in BMSC medium at two different seeding densities of approximately 2.5 × 10 ⁵ or 5.0 × 10 ⁵ per flask. Two days later, cells washed with NSC medium and 5.0 × 10 ⁵ NSC (THD-015, P14) were seeded in 15 ml of complete NSC medium on the surface of BMSC. Cells were co-cultured for 15 days, supplemented with nutrients by replacing half of the medium with fresh NSC medium every 2 or 3 days. After 15 days, cells were collected by trypsinization. Large BMSCs and small NSCs were counted on a hemocytometer. For example, in Example 2, etc., BMSC was exhausted as described elsewhere herein. The cells were resuspended to approximately 5 × 10 × 10 ⁷ / ml and incubated with anti-human CD13 antibody for 15 minutes on ice. Cells were washed once and incubated with washed pan-mouse IgG Dynabeads ™ for 30 minutes. Bead-bound cells were separated on a magnetic particle concentrator. The supernatant containing NSC was washed with NSC medium, and the number of regenerated NSCs was counted. Some of the cells were analyzed by FACS and others were subjected to differentiation and immunocytochemistry.

The disclosure herein demonstrates that NSC culture on fewer BMSC feeder cell layers causes greater NSC proliferation than many BMSCs. By changing the ratio of BMSC to NSC seeding density, when BMSC is seeded at a low confluence of about 30% compared to when BMSC is seeded at 50-60% or high confluence, it is better than that of NSC. A discovery was made that high fold growth was observed. For example, as shown in Table 3, when the BMSC is about 2.5 × 10 ⁵ and the NSC is 5.0 × 10 ⁵ for the seeding density in the T-75 flask, 47 times when the same number of NSCs are seeded on twice the BMSC. An 82-fold increase in NSC was observed compared to a 2-fold increase. In comparison, when NSCs were cultured in standard NSC medium supplemented with exogenous LIF on coated dishes without BMSC, the maximum NSC growth achieved in other experiments was approximately 20- 25 times. Following NSC growth in the presence of BMSC, BMSC is efficiently depleted with a recovery rate of 83-93% from co-culture with anti-BMSC antibody and immuno-magnetic beads (Table 3).

  In cocultures where NSCs were grown and isolated, it was observed that expression of markers of progenitor cells including, but not limited to, nestin continues (FIG. 10). The isolated NSCs also maintained the potential to differentiate into neurons and astrocytes (Figure 11). Based on the present disclosure, those skilled in the art can further scale up the method for growing NSCs and adapt it to a system similar to the manufacturing process that has become effective for the production of BMSCs. Will be able to recognize.

(BMSC and NSC co-culture in Transwell (registered trademark) (contact-independent co-culture))
It has been demonstrated herein that BMSC can serve as an excellent supply / support layer for the proliferation and expansion of human neural stem cells (hNSC). In this example, physical contact between two cell types is required or in a contact-independent manner that provides soluble trophic factors to support NSC growth and expansion, and NSC growth and Experiments were planned to solve whether the BMSC could support the expansion.

  The designated hBM-03-016-Pl and hBM-012-P2 BMSCs were thawed, washed in BMSC medium, and seeded in Transwell®. Experiments with Transwell® were performed in a Coster ™ 6-well dish. BMSCs were seeded in Transwell® while NSCs were seeded at the bottom of the hole. Both cell types were cultured in the same NSC medium. The use of Transwell® allows two cell types such as BMSC and NSC to be cultured without different physical contact with each other.

  Approximately 30,000 BMSCs were seeded on the surface of Transwell ™ removable porous filters. Overnight, the cells were allowed to adhere to the surface of Transwell ™ in BMSC medium, then the cells were washed with serum-free medium, complete NSC medium (DMEM / F12 + N2 supplement + EGF + FGF ). NSCs (designated THD-hWB-015-P13) were thawed, washed and seeded at a density of approximately 40,000 cells / well on 6-well dishes coated with polyornithine / fibronectin. Then, Transwell (trademark) containing BMSC was placed in a 6-well plate containing NSC. The control contains NSC and does not have BMSC in the transwell. Sufficient medium was added to both the transwell and the bottom of the well. About 50% of the medium was changed every other day. After 2 weeks of culture, NSCs were collected from each well and counted. Cells were analyzed by expression of NSC markers using flow cytometry.

  The results demonstrated that NSCs cultured with BMSCs in transwells in coated wells grew better than NSCs cultured without BMSCs in coated holes (FIG. 12). BMSC was observed to support NSC proliferation and expansion even in the absence of direct contact between the two cell types. Without being limited by a particular theory, it is believed that factors secreted from BMSCs supplement NSC medium and thus help support NSC growth and expansion. FACS analysis of NSCs cultured in the presence of BMSCs in transwells or NSCs cultured alone in the absence of BMSCs demonstrated that NSCs were similar in phenotype. For example, NSCs cultured under both conditions were observed to be almost 100% CD133 positive.

  The same experiment was performed except that the 6-well dish was not coated with polyornithine / fibronectin. About 50,000 BMSCs were seeded in the transwell. NSC (designated THD-hWB-015-P12) was thawed and seeded in each of the Falcon ™ and Coster ™ 6-well plates. BMSCs cultured in transwells were seeded in 6-well plates and the cultures were maintained for 2 weeks. As a control, NSCs were cultured alone in NSC medium with or without exogenous LIF. NSCs cultured with BMSCs in transwells grew in the same manner as NSCs cultured alone in NSC medium in the presence of exogenous LIF, but more than NSCs grown alone in the absence of exogenous LIF Also grew remarkably well (FIG. 13). It was also observed that with respect to the amount of cell growth, the Coster ™ dish provided better results than the Falcon ™ dish with respect to the amount of cell growth.

(NSC growth in BMSC conditioned medium)
The results of the experiment shown in Example 4 demonstrated that BMSC created a soluble factor that maintained NSC growth. To further elucidate the effects of factors secreted from BMSC on NSC proliferation, the following set of experiments was performed using conditioned media from cultured BMSCs. The experiments disclosed herein examined whether growth secreted from BMSCs and / or other factors could support and promote NSC growth and expansion. In this experiment, 1) whether the medium prepared by cultured BMSC can replace NSC medium supplemented with exogenous LIF that gives growth-promoting effect on NSC during culture, or 2) cultured BMSC It was planned to investigate whether the medium conditioned by can give the cultured NSC a beneficial effect similar to that of culturing NSCs in dishes coated with polyornithine / fibronectin.

  To produce BMSC-CM, BMSCs were first seeded in T-80 flasks and cultured in BMSC medium. After the culture period, the BMSC medium was replaced with NSC medium. As a result, BMSC-CM was obtained from BMSC cultured in either complete NSC medium (BMSC-CM1) containing bFGF and EGF or NSC medium (BMSC-CM2) not containing EGF and bFGF. BMSCs were given fresh NSC medium every 48 hours. At this stage, the media was removed, centrifuged to remove particulate matter, and used to feed NSCs or frozen at -80 ° C for cytokine testing. For BMSC-CM experiments, NSCs were given NSC medium containing approximately 25-50% BMSC-CM that had been freshly recovered from BMSC or stored at 4 ° C. for about 2 weeks. In addition, BMSC-CM was analyzed for the presence of various cytokines by cytokine arrays (trademark, manufactured by Ray Biotech) or by enzyme-linked immunoassay (ELISA).

  To test whether NSCs can be grown in BMSC-CM, NSCs were seeded in dishes coated with polyornithine / fibronectin, a) BMSC-CM1, b) BMSC-CM2, c) Nutrition was provided by replacing complete NSC medium with exogenous LIF or d) complete NSC medium without exogenous LIF with 50% medium every other day. For the first two exchanges of BMSC-CM1 and BMSC-CM2, each BMSC-CM was added with new EGF and FGF, but thereafter EGF and FGF were not added to BMSC-CM.

This result indicates that NSCs grew equally well in BMSC-CM or complete NSC medium. FIG. 14 represents a typical field of NSCs cultured under different conditions. In the presence of BMSC-CM1, it was observed that NSCs formed large adherent neurospheres with the neurospheres that allowed the cells to grow outside the neurospheres. It was also observed that some of the NSCs cultured in the presence of BMSC-CM1 formed a monolayer. In addition, when NSCs were cultured in BMSC-CM2, neurospheres formed from NSCs were observed. However, compared to NSCs cultured in the presence of BMSC-CM1, relatively few large neurospheres were formed, and relatively few monolayers appeared. In the presence of complete NSC medium, several neurospheres connected to each other to grow NSCs, indicating cells growing from the neurospheres. In either case, cells in each culture set were collected after 10 days and the number of cells was counted to compare the effect of different culture conditions on NSC growth. The number of cells recovered from BMSC-CM1 (approximately 3.4 × 10 ⁶ cells) was similar to that of complete NSC medium with exogenous LIF (approximately 3.3 × 10 ⁶ ). The number of cells obtained with BMSC-CM2 was approximately 2.85 × 10 ⁶ .

  In the next set of experiments, NSCs were tested for their ability to grow in BMSC-CM even on uncoated dishes. The four groups of NSC are as follows: 1) NSCs cultured in BMSC-CM1; 2) NSCs cultured in BMSC-CM2; 3) NSCs cultured in complete NSC medium with exogenous LIF; 4) NSCs cultured in complete NSC medium without exogenous LIF. Cells were cultured for 2 weeks, passaged by trypsinization, and treated for 2 more weeks under similar conditions. In the first 2 weeks, maximal proliferation of NSCs was observed in BMSC-CM1, followed by BMSC-CM2, and then in NSC medium + LIF (Figure 15, bar graph shown in PI). After the passage, BMSC-CM1 grew in the same manner as NSC in NSC medium + LIF (indicated as P2 in FIG. 15). Cells treated with BMSC-CM2 continued to grow and were better than cells treated with NSC medium without LIF. That is, the present data indicate that BMSC-CM is an excellent material for growth factors for hNSC and can induce NSC growth at a rate equal to or faster than NSC medium supplemented with exogenous LIF. In addition, NSCs cultured in BMSC-CM can be passaged by trypsin treatment and can be further proliferated to increase the number of cells.

(BMSC-CM cytokine array analysis)
BMSC were cultured in complete NSC medium. The medium was collected after 48 hours and the cytokine profile characteristics were determined using a cytokine assay. Ray BioHuman Cytokine Antibody Array C Series 1000 (trademark, combination of Arrays VI and VII) was purchased from Ray Biotech (Norcross, GA). Array membranes were treated with samples as described in the manufacturer's manual. Final detection of cytokines was performed using the ECL-Plus system (Trademark, Amersham, Piscataway, NJ). The signal was visualized on X-ray film (Amersham, Piscataway, NJ).

  Cytokine characterization results indicated that various cytokines / factors are present in BMSC-CM based on the intensity of the signal above the negative control on the same array. Cytokines / factors include, but are not limited to, LIF, brain-derived neurotrophic factor (BDNF), epidermal growth factor receptor (EGF), basic fibroblast growth factor (bFGF), FGF-6, glial cells Neurotrophic factor (GDNF), granulocyte colony stimulating factor (GCSF), hepatocyte growth factor (HGF), IFN-γ, insulin-like growth factor binding protein (IGFBP-2), IGFBP-6, IL-lra, IL- 6, IL-8, monocyte chemotactic protein (MCP-1), mononuclear phagocyte colony stimulating factor (M-CSF), neurotrophic factor (NT3), tissue metalloprotease inhibitor (TIMP-1) , TIMP-2, tumor necrosis factor (TNF-β), vascular endothelial growth factor (VEGF), VEGF-D, urokinase plasminogen activator receptor (uPAR), bone morphogenetic protein 4 (BMP4), IL1- a, IL-3, leptin, stem cell factor (SCF), stromal cell derived factor-1 (SDF-1), platelet derived growth factor-BB (PDGFBB), tumor cell growth factor beta (TGFβ-1), and TGFβ- Including 3.

  While not being limited to a particular theory, in addition to factors secreted by BMSC or induced by the medium, several factors, including those already present in the medium, including EGF and bFGF, are -CM includes. Factors are exemplified herein based on an evaluation of the signal above the negative control of an array of 120 cytokines present in the tested array.

  Some of the cytokines detected above background (baseline) levels include, but are not limited to, EGF and bFGF. Other apparent cytokines are HGF (hepatocyte growth factor), M-CSF (macrophage colony stimulating factor), and TIMP-1 and TIMP-2 (tissue metalloprotease inhibitors 1 and 2, respectively). Neurotrophic factors include, but are not limited to, BDNF, NT3, GDNF, and CNTF. IGFBP and FGF-6 were present. Many chemokines, inflammatory cytokines and angiogenic factors such as VEGF have also been produced. BMSC, also confirmed by ELISA, produced LIF. In the ELISA assay, the actual amount of each secreted factor from the BMSC tested was calculated by subtracting the control medium baseline level from the value measured with BMSC-CM.

  From the disclosure presented herein, it can be concluded that BMSCs produce soluble factors that promote NSC growth. Also, direct contact of NSCs with BMSCs is not necessary because conditioned BMSCs can support NSC growth and expansion. The present disclosure shows that contact-dependent co-culture elicits the highest NSC growth, which is the NSC growth synergistic effect by contact with BMSC or BMSC products and soluble factors produced by BMSC along with NSC. It indicates what is suggested.

(NSCs cultured on or in BMSC-CM show a decreased expression level of MHC molecules, but maintain similar phenotypic characteristics as NSCs cultured alone under standard conditions.)
The NSCs grown in Example 3 were analyzed by flow cytometry for the expression of various BMSCs and NSC markers. NSC co-cultured with BMSC is non-BMSC in NSC medium supplemented with exogenous LIF, except that NSC co-cultured with BMSC does not show detectable expression of MHC class II molecules Maintain the same phenotypic characteristics (eg, CD56 and CD133 positive and CD105 negative) as NSC cultured in (FIG. 16). When exogenous LIF was added to NSC medium, it was observed that MHC class II molecules were induced by NSC. In contrast, NSCs co-cultured with BMSCs have no observed expression of MHC class II molecules beyond baseline, which can favor transplantation. Furthermore, NSCs isolated from co-culture did not express BMSC marker CD105 beyond baseline, indicating that BMSCs were efficiently used up from co-culture.

  Similarly, NSCs cultured in BMSC-CM have the same phenotypic characteristics as NSCs cultured in standard NSC medium + LIF, except that baseline values show a decrease in MHC class II and MHC class I (Fig. 17). Four groups of NSCs were FACS analyzed for expression of CD133, MHC class II molecules, MHC class I molecules, and CD56 (FIG. 17). The results confirmed previous observations that NSCs cultured in the presence of exogenous LIF expressed MHC class II molecules and also showed increased median fluorescence of MHC class I molecules. However, NSCs grown on BMSC-CM showed no expression of MHC class II molecules, and the level of MHC class I molecule expression was low. CD133 expression was also the same in all cases. All cells were tested for expressed CD56, but cells cultured in the presence of exogenous LIF showed a decrease in CD56 median fluorescence.

(Control of MHC class I and class II expression of NSCs grown on BMSC)
Additional experiments were performed to evaluate the expression of both MHC class I and class 11 molecules by co-cultured NSCs. NSCs were isolated from fetal brain and cultured for 13 passages in NSC medium (THD-WB-015, P13). Next, NSCs were cultured under the following four conditions for 12 days, changing the medium every other day:
1. 62,500 NSCs alone in complete NSC medium in a coated T25 flask.
2. Single NSC 62,500 in complete NSC medium + LIF in a coated T25 flask.
3. 62,500 NSCs in complete NSC medium on 250,000 BMSC (donor-012) feeder cell surface.
4. NSC in complete NSC medium on 250,000 BMSC (donor-016) feeder cell surface.

  After 12 days, cells were harvested by trypsinization. Cells were counted and the expression of CD133, CD105, MHC class I and MHC class I was analyzed by flow cytometry. In the case of co-culture, all cells were obtained and NSCs were gated based on light scattering and CD133 expression. FIG. 18 shows the expression of MHC class I and class II molecules by NSCs cultured under various conditions (black = isotype control; gray = class I or class II).

  The results indicate that all NSCs express MHC class I. However, when NSCs were cultured in the presence of exogenous LIF, as indicated by the increase in log median fluorescence intensity when compared to NSCs cultured in the absence of exogenous LIF, MHC class I in the cells The expression of was significantly increased. Compared to NSCs grown with LIF, co-cultured NSCs expressed MHC class I at significantly lower levels (Table 4).

  NSC did not constitutively express MHC class II molecules. On the other hand, LIF induced the expression of MHC class II molecules in 38% of the cells. NSCs cultured with BMSC, like NSCs cultured in the absence of LIF, did not express MHC class II beyond baseline levels.

  From the above results, the benefits of growing NSCs on BMSCs can reduce and / or suppress the expression of NSC immunoregulatory MHC class I and II molecules, making them more suitable for clinical transplantation. Indicated.

  The disclosures of all patents, patent applications, and publications cited herein are generally used herein as material.

  It will be apparent to those skilled in the art that various modifications and variations can be made in the method and structure of the present invention without departing from the spirit or scope of the invention. That is, the present invention is intended to cover the scope of modifications and variations of the present invention which fall within the scope of the appended claims and their equivalents.

It is a graph which shows the proliferation of BMSC in a BMSC culture medium. 2A shows NSC neurospheres growing in the absence of exogenous LIF. 2B and 2C show NSCs grown in the form of neurospheres in the presence of exogenous leukemia inhibitory factor (LIF). 3A and 3B are images showing BMSC and NSC co-culture in the absence of exogenous LIF. 3C and 3D are images showing BMSC and NSC co-culture in the presence of exogenous LIF. 4A-4F are pre-differentiation images showing nestin and glial fibrillary acidic protein (GFAP) expression by NSCs co-cultured with BMSC. 4G and 4H indicate the absence of nestin and GFAP by BMSC cultured alone. It is an image which shows that NSC propagated on BMSC possesses the ability to differentiate into a neuron | cell and an astrocyte. FIG. 5 is an image showing nestin-GFAP staining of a differentiated co-culture showing very little nestin expression after differentiation. It is an image showing MAP2-GFAP staining of differentiated NSC. 8A and 8B are images showing MAP2-GFAP-DAPI staining of differentiated BMSCs that show negligible expression of neuronal and astrocyte markers. 9A is a FACS analysis graph showing the phenotype of NSC after removal of BMSC, indicating that less than 2% of cells are CD-105 (BMSC marker) positive. 9B is a FACS analysis graph showing the NSC phenotype after removal of BMSC, indicating that more than 90% of the cells are CD133 positive. 10A is a FACS analysis graph showing expression in the absence of nestin by BMSC. FIG. 10B is a FACS analysis graph showing nestin expression by NSCs isolated from co-culture using BMSC feeder cells. It is an image showing that NSCs proliferating on BMSC possess pluripotency to differentiate into neurons and astrocytes. It is a graph which shows the proliferation of NSC in Transwell (trademark) in the presence or absence of BMSC. 13A is an image showing NSC grown on an uncoated plate in the presence of BMSC in Transwell ™. 13B is an image showing NSC grown on an uncoated plate in the absence of BMSC in Transwell ™. 14A is an image showing NSC cultured using bone marrow stromal cell conditioned medium (BMSC-CM). 14B is an image showing NSC cultured using bone marrow stromal cell conditioned medium (BMSC-CM). 14C is an image showing NSC cultured in NSC medium in the presence of exogenous LIF. 14D is an image showing NSCs cultured in NSC medium in the absence of exogenous LIF. FIG. 6 is a graph showing the effect of BMSC-CM compared to standard NSC medium with or without exogenous LIF on NSC growth. 16A is a FACS analysis graph showing the phenotypic profile of NSCs isolated from co-cultures using BMSCs from two different donors. 16B is a FACS analysis graph showing the phenotypic profile of NSCs isolated from co-cultures using BMSCs from two different donors. 16C is a graph showing the phenotype profile of NSCs cultured in NSC medium without BMSC. 16D is a graph showing the phenotypic profile of NSCs cultured in NSC medium with exogenous LIF. 17A is a FACS analysis graph showing the phenotype of NSCs cultured in NSC medium in the presence and absence of exogenous LIF, and NSCs cultured in BMSC-CM in the presence and absence of growth factors. Yes, CD56 profile. 17B is a FACS analysis graph showing the phenotype of NSCs cultured in NSC medium in the presence and absence of exogenous LIF, and NSCs cultured in BMSC-CM in the presence and absence of growth factors. Yes, showing the profile of CD133. 17C is a FACS analysis graph showing the phenotypes of NSCs cultured in NSC medium in the presence and absence of exogenous LIF, and NSCs cultured in BMSC-CM in the presence and absence of growth factors. Yes, showing the profile of MHC class II molecules. 17D is a FACS analysis graph showing the phenotype of NSC cultured in NSC medium in the presence and absence of exogenous LIF, and NSC cultured in BMSC-CM in the presence and absence of growth factors. Yes, showing the profile of MHC class I molecules. 18A is a FACS analysis graph showing the phenotype of NSCs cultured in NSC medium in the absence of exogenous LIF, showing the expression of MHC class I and class II molecules by NSCs cultured under various conditions. 18B is a FACS analysis graph showing the phenotype of NSCs cultured in NSC medium in the presence of exogenous LIF, showing the expression of MHC class I and class II molecules by NSCs cultured under various conditions. 18C is a FACS analysis graph showing the phenotype of NSC co-cultured with BMSC in complete NSC medium and shows the expression of MHC class I and class II molecules by NSCs cultured under various conditions. 18D is a FACS analysis graph showing the phenotype of NSC co-cultured with BMSC in complete NSC medium, showing the expression of MHC class I and class II molecules by NSCs cultured under various conditions.

Claims (43)

  A composition comprising (a) an isolated bone marrow stromal cell (BMSC), and (b) a chemically defined medium containing a neural stem cell (NSC) growth medium and a factor secreted by the BMSC.   The composition of claim 1, wherein the medium does not contain exogenous leukemia inhibitory factor (LIF).   The composition of claim 1, wherein said factor is selected from the group consisting of growth factors, nutritional factors, and cytokines.   The factors include LIF, brain-derived neurotrophic factor (BDNF), epidermal growth factor receptor (EGF), basic fibroblast growth factor (bFGF), FGF-6, glial cell-derived neurotrophic factor (GDNF), granule Sphere colony stimulating factor (GCSF), hepatocyte growth factor (HGF), IFN-γ, insulin-like growth factor binding protein (IGFBP-2), IGFBP-6, IL-1ra, IL-6, IL-8, monocytes Chemotaxis protein (MCP-1), mononuclear phagocyte colony stimulating factor (M-CSF), neurotrophic factor (NT3), tissue metalloprotease inhibitor (TIMP-1), TIMP-2, tumor necrosis factor (TNF-β), vascular endothelial growth factor (VEGF), VEGF-D, urokinase plasminogen activator receptor (uPAR), bone morphogenetic protein 4 (BMP4), IL1-a, IL-3, leptin, Claim selected from the group consisting of stem cell factor (SCF), stromal cell derived factor-1 (SDF-1), platelet derived growth factor-BB (PDGFBB), tumor cell growth factor beta (TGFβ-1), and TGFβ-3 Item 2. The composition of Item 1.   The composition of claim 1 further comprising isolated NSC.   6. The composition of claim 5, wherein the NSC is in physical contact with the BMSC.   6. The composition of claim 5, wherein the NSC is not in physical contact with the BMSC.   6. The composition of claim 5, wherein the NSC is derived from the human central nervous system.   The composition of claim 1, wherein the BMSC is derived from a human.   6. The composition of claim 5, wherein exogenous genetic material has been introduced into the NSC.   The composition of claim 1, wherein exogenous genetic material is introduced into the BMSC.   Bone marrow stromal cell conditioned medium (BMSC-CM) containing neural stem cell (NSC) growth medium and a chemically defined medium containing factors secreted by isolated BMSCs.   The BMSC-CM of claim 12, wherein the BMSC-CM does not contain exogenous LIF.   The BMSC-CM of claim 12, wherein the factor is selected from the group consisting of a growth factor, a trophic factor, and a cytokine.   The factors include LIF, brain-derived neurotrophic factor (BDNF), epidermal growth factor receptor (EGF), basic fibroblast growth factor (bFGF), FGF-6, glial cell-derived neurotrophic factor (GDNF), granule Sphere colony stimulating factor (GCSF), hepatocyte growth factor (HGF), IFN-γ, insulin-like growth factor binding protein (IGFBP-2), IGFBP-6, IL-1ra, IL-6, IL-8, monocytes Chemotaxis protein (MCP-1), mononuclear phagocyte colony stimulating factor (M-CSF), neurotrophic factor (NT3), tissue metalloprotease inhibitor (TIMP-1), TIMP-2, tumor necrosis factor (TNF-β), vascular endothelial growth factor (VEGF), VEGF-D, urokinase plasminogen activator receptor (uPAR), bone morphogenetic protein 4 (BMP4), IL1-a, IL-3, leptin, Claim selected from the group consisting of stem cell factor (SCF), stromal cell derived factor-1 (SDF-1), platelet derived growth factor-BB (PDGFBB), tumor cell growth factor beta (TGFβ-1), and TGFβ-3 Item 12 BMSC-CM.   A method of modulating the expression of a major histocompatibility complex (MHC) molecule in an isolated NSC, comprising co-culture of cells comprising the isolated BMSC and the isolated NSC.   17. The method of claim 16, wherein the cells are co-cultured in the absence of exogenous LIF.   The method of claim 16, wherein the NSC is in physical contact with the BMSC.   The method of claim 16, wherein the NSC is not in physical contact with the BMSC.   17. The method of claim 16, wherein the NSC is derived from a human central nervous system.   17. The method of claim 16, wherein the BMSC is derived from a human.   17. The method of claim 16, wherein exogenous genetic material has been introduced into the NSC.   17. The method of claim 16, wherein exogenous genetic material has been introduced into the BMSC.   A method for regulating the expression of MHC molecules in isolated NSCs, comprising culturing the NSCs using bone marrow stromal cell conditioned medium (BMSC-CM), wherein the BMSC-CM comprises an NSC growth medium and A method comprising a factor secreted by said BMSC.   25. The method of claim 24, wherein the BMSC-CM does not include exogenous LIF.   25. The method of claim 24, wherein the BMSC-CM is essentially free of BMSC.   25. The method of claim 24, wherein the factor is selected from the group consisting of growth factors, trophic factors, and cytokines.   The factors include LIF, brain-derived neurotrophic factor (BDNF), epidermal growth factor receptor (EGF), basic fibroblast growth factor (bFGF), FGF-6, glial cell-derived neurotrophic factor (GDNF), granule Sphere colony stimulating factor (GCSF), hepatocyte growth factor (HGF), IFN-γ, insulin-like growth factor binding protein (IGFBP-2), IGFBP-6, IL-1ra, IL-6, IL-8, monocytes Chemotaxis protein (MCP-1), mononuclear phagocyte colony stimulating factor (M-CSF), neurotrophic factor (NT3), tissue metalloprotease inhibitor (TIMP-1), TIMP-2, tumor necrosis factor (TNF-β), vascular endothelial growth factor (VEGF), VEGF-D, urokinase plasminogen activator receptor (uPAR), bone morphogenetic protein 4 (BMP4), IL1-a, IL-3, leptin, Claim selected from the group consisting of stem cell factor (SCF), stromal cell derived factor-1 (SDF-1), platelet derived growth factor-BB (PDGFBB), tumor cell growth factor beta (TGFβ-1), and TGFβ-3 Item 24. The method according to Item 24.   25. The method of claim 24, wherein the NSC is derived from a human central nervous system.   25. The method of claim 24, wherein exogenous genetic material has been introduced into the NSC.   An isolated NSC produced by a co-culture method of isolated BMSC and isolated NSC cells.   32. The isolated NSC of claim 31, wherein expression of said NSC MHC class I molecule is reduced.   32. The isolated NSC of claim 31, wherein said NSC MHC class II molecule exhibits a baseline level.   32. The isolated NSC of claim 31, wherein said NSC is derived from the human central nervous system.   32. The isolated NSC of claim 31, wherein exogenous genetic material has been introduced into the NSC.   An isolated NSC produced by culturing an isolated NSC with BMSC-CM, wherein the BMSC-CM is a chemically defined medium containing an NSC growth medium and a factor secreted by the isolated BMSC. Contains isolated NSC.   37. The isolated NSC of claim 36, wherein expression of said NSC MHC class I molecule is reduced.   37. The isolated NSC of claim 36, wherein said NSC MHC class II molecule exhibits a baseline level.   37. The isolated NSC of claim 36, wherein said NSC is derived from the human central nervous system.   37. The isolated NSC of claim 36, wherein exogenous genetic material has been introduced into the NSC. (a) Isolated NSC
(b) Isolated BMSC
(c) an NSC growth medium containing factors secreted from the isolated BMSC, and
(d) A nerve cell culturing apparatus including means for preventing the NSC and the BMSC from coming into physical contact with each other.   42. The apparatus of claim 41, further comprising a filter or membrane to prevent the NSC and the BMSC from being in physical contact with each other.   43. The device of claim 42, wherein the filter or membrane has pores that allow factors secreted from the BMSC to permeate the filter or membrane.

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